(1) Field of the Invention
The present invention relates to a method for producing novel monoclonal antibodies against a trichothecene and to a test kit and method which use these monoclonal antibodies to detect the presence of a trichothecene in foods and the like. In particular the present invention relates to monoclonal antibodies produced by repeated introduction, preferably subcutaneously, of a trichothecene polypeptide into a murine over a period of time so that polyclonal antibodies are developed in the blood serum of the murine and then by the production of hybridomas from spleen cells of the murine which generate the monoclonal antibodies.
(2) Prior Art
Mycotoxins are chemical substances produced by fungi which affect the health of humans and animals. The fungi are not involved in the disease process and are often absent from the food source at the time of poisoning. Mycotoxins are usually quite stable and remain active after processing and cooking. Mycotoxins or their metabolic products appear as residues in meat, milk and eggs making them unfit to eat.
The aflatoxins are probably the best known mycotoxins because of their mutagenic and carcinogenic properties. The fluorescent properties of the aflatoxins make detection fairly easy. Recently, a group of mycotoxins known as the trichothecenes have become the focus of attention because of their detection in corn and small grains, and because the trichothecenes may have been used for chemical warfare. The trichothecenes are difficult to identify because they do not contain a functional group which permits easy identification by spectrophotometric methods, and they do not react specifically with any reagent which provides a colorimetric assay. Thin layer chromatography is frequently used to detect trichothecenes, but this method is relatively insensitive and semi-quantitative due to difficulty in uniformly spraying the plates. Gas liquid chromatography (GLC), fluorodensitometry and GLC-mass spectroscopy (GL-MS) have been used successfully but do not lend themselves to application in the field. Only GL-MS will positively identify a suspected trichothecene. These procedures are also time consuming and expensive.
Trichothecenes are produced by several species of fungi, but most predominantly by Fusarium sp. There are at least 37 known natural trichothecene derivatives, six of which are somehow involved in Fusarium toxicosis.
The trichothecenes, when applied to the shaved skin of a rat or a rabbit, produce a strong dermititic reaction. This crude bioassay is sensitive but not specific. The reaction is characterized by severe local irritation, inflammation, desquamation, subepidermal hemorrhaging, and general necrosis. When given orally or by intraperitoneal injection the trichothecenes are acutely toxic at low concentrations. For example, T-2 toxin in rats has an LD.sub.50 of 3.0 mg/Kg body weight when administered by intraperitoneal injection, and 3.8 mg/Kg when administered orally.
General symptoms of test animals orally dosed with trichothecenes are listlessness, development of diarrhea, and rectal hemorrhaging. Necrotic lesions can develop in the mouth parts. The mucosal epithelium of the stomach and small intestines erode, accompanied by a hemorrhage which can develop into a severe case of gastrointestinitis, followed by death. The cells of the bone morrow, lymph nodes, and intestines undergo a pathological degeneration. In large animals, massive hemorrhages develop in the lumen of the small intestines. The trichothecenes are also more potent protein inhibitors than the antibiotics puromycin and cycloheximide. The effects of ingesting sub-lethal doses of the trichothecenes over a long period of time are not known. One of the trichothecenes, T-2, is believed responsible for alimentary toxic aleukia (ATA) which caused thousands of deaths in Russia (1944-47) when Fusarium infected grain was ingested.
Several immunological studies have been done on mycotoxins (Chu, F. S., S. Grossman, Ru-Dong Wei, and C. Mirocha. Applied and Environmental Microbiology 37:104-108 (1979); Lee, S. and F. S. Chu. J. Assoc. Off. Anal. Chem. 64:156-161 (1981); and Lee, S. and F. S. Chu. J. Assoc. Off Anal Chem 64:684-688 (1981)). Highly specific polyclonal antibodies were produced in rabbits to a bovine serum albumin (BSA) conjugate to Ochratoxin A, BSA conjugates to Aflatoxins B.sub.1, B.sub.2, and M.sub.1 derivatives, and a BSA conjugate to a T-2 derivative. Specific polyclonal antibodies were also produced in rats and goats immunized with an aflatoxin B.sub.1 -BSA conjugate but titer levels were lower than in the analogous antisera from rabbits. A highly sensitive radioimmunoassay (RIA) was developed for these systems. Using polyclonal antibodies produced to aflatoxin B.sub.1 and ochratoxin A, a sensitivity of 5-10 ppb was obtained with crude corn, wheat, urine and blood extracts.
It was therefore decided that it would be advantageous to use monoclonal antibodies for conducting the tests. This would provide an advantage over more traditional serological techniques due to the possibility that hybridomas could produce unlimited amounts of highly uniform monoclonal antibodies with specificities comparable with the best rabbit antiserum. These monoclonal antibodies could then be used in the development of a colorimetric commercial assay system for the mycotoxins, such as Enzyme Linked Immunoabsorbant Assay (ELISA) or fluorescent antibody tests.